It has been postulated that Acquired Immune Deficiency Syndrome (AIDS), AIDS related complex (ARC) and pre-AIDS are caused by a retrovirus, the Human Immunodeficiency virus type 1 HIV-1; also known as HTLV-III, LAV-1 and ARV). Recently another pathogenic human retrovirus named HIV-2 (formerly LAV-2) was isolated from west African patients with AIDS (Montagnier et al, in PCT/FR 87/00025, published on Jul. 30, 1987 under International Publication no. WO 87/04459). It has recently been shown (Guyader et al. Nature 326, 662-669, 1987) that HIV-2 shares a number of conserved sequences with HIV-1 and the Simian Immunodeficiency viruses (SIV).
Even though other numbering systems are used in the prior art referred to herein, the numbering systems for amino acids used herein is that of Ratner et al., Nature, 313 277-284, 1985 for the HIV-1 proteins and that of Guyader et al, Nature, 326, 662-669 (1987) for the HIV-2 proteins. The amino adds used herein in the peptides are given with the single letter code as follows: ala=A, arg=R, asn=N, asp=D, cys=C, gln=Q, glu=E, gly=G, his=H, ile=l, leu=L, lys=K, met=M, phe=F, pro=P, ser=S, thr=T, trp=W, tyr=Y and val=V.
The initial immunodiagnostic tests for the detection of antibodies in the serum of patients infected with HIV-1 utilized the whole virus as antigen. Second generation tests made use of polypeptide sequences obtained by the recombinant DNA methodology. Cabradilla et al. Bio/Technology 4 128-133 (1985) and Chang et al. Bio/Technology 3, 905-909 (1985) succeeded in obtaining bacterially synthesized viral protein fragments of 82 and 102 amino acid residues respectively. E.P. 86202314 and 86114243 describe recombinant polypeptides covering regions of the gp41 and gp120 that are immunoreactive alone or in mixtures. Shoeman et al. Anal. biochem. 161, 370-379 (1987) also describe several polypeptides from gp41 that have immunoreactive properties with antibodies present in sera from patients infected with HIV-1. None of the above assay procedures is acceptable. Their lack of sensitivity is serious as it may permit blood containing virus to escape detection and thereby potentially res the infect blood product receivers. The impurities present in these antigen preparations are also responsible for unacceptably high levels of false positive results which cause healthly individuals to suffer distress.
It then became apparent that a tendency of the prior art was the identification of shorter epitopes. This is because of the ease and lower cost with which they could be prepared and more importantly because of the reduced risk of obtaining falsely positive test results due to the presence of shared epitopes with viral proteins not related to AIDS. In this regard, Gallaher, (Cell 50, 327-328, 1987) has found that a region of the gp41 of HIV-1 shares a sequence of five adjacent amino acid residues with the respiratory syncytial virus and of four equally distributed amino acids of the measles virus F1 glycoprotein. Thus, even highly purified recombinant polypeptides containing this region, or any other common regions yet to be discovered, would potentially be responsible for falsely positive results.
Apart from its superior specificity, the identification of shorter peptide sequences corresponding to unique and highly conserved epitopes of the HIV viruses makes its production by chemical synthesis easier and cheaper. Empirical methods have been described. These methods are capable of assisting in the selection of short amino acid sequences which are likely to be exposed on the surface of the native protein (for a review see Hopp and Woods, J. Immunol. Met. 88: 1-18, 1986). Although somewhat useful, these methods are no more than indicative. Nonetheless they have been applied by many for the identification of epitopes present on the protein of the viruses responsible for AIDS. For example: U.S. Pat. No. 4,629,783, International Patent Appl. No. PCT/US86/00831. and E.P. Appl. No. 86303224 disclose various synthetic peptides from the p18, p25, gp41 and gp120 proteins of HIV-1 that are claimed useful in AIDS diagnostic kits.
This trend towards smaller antigens however is accompanied by a risk that the synthesized epitope is not able to assume a rigid conformation that is recognized by the antibody. Although the number of serum samples tested in each of these cases is very limited, specificity was found to be very high (95%-100%) with small synthetic peptides but the overall sensitivity varied between 80 and 100%. In the only example where 100% sensitivity was attained only ten samples had been tested.
Smith et al., (J. Clin. Microbiol. 25, 1498-1504, 1987) described two overlapping peptides, E32 and E34, that are highly immunoreactive. No false positive result, out of 240 seronegative specimens, were obtained but the test missed three seropositive samples out of 322 (sensitivity of 99.1%). Wang et al. (Proc. Natl. Acad. Sci. 83, 6159-6163, 1986) described a series of overlapping peptides (including amino acid residues of the E32 and E34 peptides discovered by Smith et al.) among which one 21-mer peptide showed 100% specificity and 98% sensitivity (out of 228 seropositive samples taken from patients with AIDS, 224 were found positive with this peptide).
In U.S. patent appl. Ser. No. 120,027 Filed Nov. 13, 1987, there is disclosed a short synthetic peptide covering residues 606 to 620 (SGKLICTTAVPWNAS) of gp41 (HIV-1) which is said to be immunoreactive with antibodies of patients infected by the AIDS viruses. In this example, specificity was also excellent (63/63) but 6 seropositive specimens out of 57 confirmed positive could not be detected (sensitivity of 89%).
Gnann et al. (J. Virol. 61, 2639-2641, 1987 and J. Infec. Dis. 156, 261-267, 1987) also reported a series of overlapping peptides from an immunodominant region of gp41 (HIV-1). Of particular interest was their finding that one peptide having the sequence SGKLIC (606-611) was not immunoreactive with any of the 22 HIV-1 positive sera tested. The addition of a cysteine residue to the N-terminus restored some immunoreactivity, 21 of 44 sera reacted with the 7-mer peptide (48% sensitivity). Gnann et al. concluded that cys-605 was essential for the immunoreactivity of that segment of the gp41-(HIV-1) protein.
Gnann et al. have also speculated that the cysteine residues at positions 605 and 611 (Ratner's numbering system) of gp41 (HIV-1) might play a critical role in the antigenic conformation of this region of the protein possibly through the formation of a loop via disulfide bonding. However, attempts by the authors to identify and prove the formation of the disulfide bonding have failed. Since Gnann et al. never demonstrated that they did have a synthetic peptide containing the partial amino acid sequence 605-611 wherein the two terminal cysteine groups were linked by disulfide bonds, the properties of a peptide having such a disulfide bond are unknown and unpredictable.
The 7-amino acid partial sequence containing two cysteine residues at position 605-611 also has been disclosed in other documents such as PCT/US 86/00831 published on Nov. 6, 1986 under International Publication No. WO 86/06414 where peptide X(39), which is encoded by the region from about bp 7516 through 7593, and peptide XIII(79) which is encoded by the region extending from about bp 7543 through bp 7593, both contain the 7-amino acid sequence (amino acids 605-611) discussed by Gnann et al. in the above noted publication. The peptides are reported as linear and the authors have not mentioned any formation of cyclic structures.
Rosen et al. in PCT/US 87/00577 published on Oct. 8, 1987 under International Publication No. WO 87/06005 have reported that a series of synthetic peptides encompassing the Cys(605)-Cys(611) residues of the HIV-1 envelope glycoprotein (gp41) undergo a series of spontaneous oxidative transformations upon solubilization in neutral or basic aqueous buffer. The authors have speculated that under these conditions, the peptides used in ELISAs are a random mixture of linear monomer, cyclic monomer, linear or cyclic dimers and linear polymers of various lengths. However, the inventors did not prove the presence of cyclic components and have not characterized the other various dimers and polymers present, they have speculated that the polymer forms are the most important components for reactivity in ELISA testing.
Gnann et al. (Science 237, 1346-1349, 1987) reported a short linear synthetic peptide covering residues 592 to 603 of gp42 (HIV-2) that contains two cysteines in a region homologous to the one on gp41 (HIV-1) including Cys(605) and Cys(611). This peptide reacted with 5 out of 5 sera taken from HV-2 infected patients.
Although the references discussed above do provide peptides which are useful in identifying HIV-1 antibodies, they also present certain drawbacks such as inability to full detection (100%) of positive serum samples. For example, Gnann et al. (J. Virol. 61, 2639-2641, (1987)) in their tests with their 600-611 amino acid sequence detected 22 out of 22 positive sera however they also stated that similar tests carried out by another author at the Centers for Disease Control, Atlanta, Ga. with the same 12-amino add sequence (600-611) detected 78 out of 79 positive sera. Gnann et al. in J. Infect. Dis., 156, 261-267, 1987 showed that the same 12-amino acid sequence from gp41-(HIV-1) was shown to be reactive with 131 out of 132 HIV-1 infected patients from the United States.
In the same article, it is also clearly shown that when the HIV-1 positive sera are diluted by a factor exceeding 500, some of these diluted sera are found to be negative thus indicating a low sensitivity.
Another potential drawback of these prior art assays is their use of a poorly defined and unpredictable peptide mixture as the probe. This mixture comprises peptides having many oxidative forms of cysteine produced spontaneously during peptide preparation, processing and use.
It would appear highly desirable to provide peptides or peptide mixtures which are resistant to spontaneous oxidation. Such peptides would, thus, have a well defined structure. Moreover, such peptides would, under normal test conditions, detect all HIV-1 and/or HIV-2 antibody-containing samples as positive even when extremely low levels of antibody are present.